Poly(hydroxyalkanoates) (PHAs) are well-known polyester compounds produced by a variety of microorganisms, such as bacteria and algae. A PHA polyester can include the same or different repeating units, depending upon the choice of carbon source substrates and fermentation conditions employed in the production of the PHA. One particular PHA including the same repeating units is poly(3-hydroxybutyric acid), or poly(3-hydroxybutyrate), termed PHB, and having the structural formula: ##STR2## PHB is a natural storage product of bacteria and algae, and is present as discrete granules within the cell cytoplasmic space. However, unlike other biologically-synthesized polymers such as proteins and polysaccharides, PHB is thermoplastic having a high degree of crystallinity and a well-defined melting point at about 180.degree. C. But, PHB is unstable at its melting point and degrades, essentially quantitatively, to crotonic acid at a temperature slightly above its melting point. Accordingly, practical applications for this natural, biodegradable polyester have been limited. Therefore, investigators have studied other PHAs, such as the biodegradable copolyester poly(hydroxybutyrate-co-valerate), including both of the monomeric units 3-hydroxybutyrate and 3-hydroxyvalerate, in order to discover a PHA having sufficient thermal stability and other suitable chemical and physical properties for use in practical applications.
Generally, a PHA is synthesized by a microorganism. However, some PHA compounds have been synthesized chemically, such as by polymerization of racemic and optically-active butyrolactone or other suitable monomers. Such chemically-synthesized PHA polyesters exhibited a relatively low average molecular weight, and the synthesis was not economically viable. In general, the following publications provide background information for PHA polymers, both in regard to their synthesis and their properties:
1) E. A. Dawes, et al., Adv. Microb. Physiol., 10, p. 135 (1973);
2) P. A. Holmes, "Developments in Crystalline Polymers-2", D. C. Basset, ed., Elsevier Applied Science, London, Chap. 1, pp. 1-65 (1988); and
3) P. A. Holmes, Phys Technol., 16, pp 32-36 (1985).
The preparation, extraction and purification, of a PHA by a biosynthetic process is known. For example, Richardson in European Patent Application Serial No. 046,344, and Lafferty et al. in U.S. Pat. No. 4,786,598, disclose the preparation of poly-D-(-)-3-hydroxybutyric acid (PHB) by culturing the microorganism Alcaligenes latus or a mutant thereof. Walker et al., in U.S. Pat. No. 4,358,583, teach the extraction and purification of poly(3-hydroxybutyric acid) from the cell walls of PHB-producing microorganisms. Furthermore, the bacterial synthesis of various co-poly(hydroxyalkanoates), such as the copolymer of 3-hydroxybutyric acid and 3-hydroxypentanoic acid, is described in publications such as:
Y. Doi, et al., "Production of Copolyesters of 3-Hydroxybutyrate and 3-Hydroxyvalerate by Alcaligenes eutrophus from Butyric and Pentanoic Acids", Appl. Microbiol. Biotechnol., 28, pp. 330-334 (1988);
Doi, U.S. Pat. No. 4,876,331;
P. Holmes, Phys. Technol., 16, pp. 32-36 (1985);
M. Kunioka,-et al., "Crystalline and Thermal Properties of Bacterial Copolyesters: Poly(3-Hydroxybutyrate-co-3-hydroxyvalerate) and Poly(3-Hydroxybutyrate-co-4-hydroxybutyrate)", Macromolecules, 22, pp. 694-697 (1989); and
R. Gross, et al., "Biosynthesis and Characterization of Poly(.beta.-Hydroxyalkanoates) Produced by Pseudomonas oleovorans", Macromolecules, 22, pp. 1106-1115 (1989).
The above-listed patents and publications are representative of the state of the art relating to PHAs. In general, the homopolymeric and copolymeric PHAs described in the above references are attempts to improve the physical and chemical properties of the PHA by altering the carbon source for the biological synthesis of the PHA, or are attempts to find a suitable microorganism to produce a sufficient amount of the desired PHA. In general, a poly(hydroxyalkanoate) has the general structural formula (I), wherein R is hydrogen or an alkyl group, and the term "a" is the number of repeating units. As illustrated in general structural formula (I), a PHA is a polyester having a hydroxy-terminated end and a carboxy-terminated end. The most widely-known and intensively-studied ##STR3## poly(hydroxyalkanoate) is the previously-described, biodegradable PHA known as poly(hydroxybutyrate), or PHB, wherein the R substitutent in general structural formula (I) is methyl. However, PHAs having an R substituent of up to nine carbon atoms have been biosynthesized and studied, as have PHAs including 4-hydroxybutyrate [(--CH.sub.2 CH.sub.2 CH.sub.2 CO.sub.2 --).sub.n ] as a repeating unit.
In addition, copolymers of general structural formula (II) have been biosynthesized by the appropriate choice of carbon substrates. For example, the copolymer of general structural formula (II), wherein ##STR4## R.sub.4 is methyl and R.sub.5 is ethyl, known as poly(hydroxybutyrate-co-valerate) or (P[HBcoHV]), has been biosynthesized and studied. In general, the copolyesters of general structural formula (II) wherein the substituents R.sub.4 and R.sub.5, independently, are hydrogen or an alkyl or alkenyl group including up to nine carbon atoms are known. Alkenyl-branched PHA's are described by K. Fritzsche, in "Production of Unsaturated Polyesters by Pseudomonas oleovorans", Int. J. Biol. Macromol., Vol. 12, pp. 85-91 (1990). In addition, a terpolymer of structural formula (III) has been biosynthesized by the bacterium Rhodospirillum rubrum from a carbon source including 3-hydroxybutyric acid, 3-hydroxypentanoic acid and 4-pentenoic acid. This terpolymer is described by R. Gross et al. in the publication, "The Biosynthesis and Characterization of New Poly(.beta.-Hydroxyalkanoates)", in Polymer Preprints, 30(1), pp. 492-493 (1989).
The biologically-synthesized PHAs exhibit a molecular weight of up to about 1,500,000 daltons. These high molecular weight, biologically-synthesized PHAs can be degraded, or depolymerized, to yield a PHA having a molecular weight as low as about 3000 daltons. For example, Trathnigg et al., in Angew. Macromol. Chem., 161, p. 1-8 (1988), described the preparation of a low molecular weight PHB by a controlled acid hydrolysis of a high molecular weight, biologically-synthesized PHB using aqueous formic, acetic or butyric acid at an elevated temperature of 90.degree.-100.degree. C. Similarly, B. Heuttecoeur, et al., in C. R. Hebd. Seances Acad. Sci., 274, pp. 2729-2732, (1972), describe the partial alkaline degradation of PHB, and S. Akita, et al., in Macromolecules, 9, pp. 774-780 (1976), describe the alcoholysis of PHB with methanol and p-toluenesulfonic acid. The methods of Trathnigg and of Heuttecoeur provide a degraded PHB polymer with a carboxylic acid or a carboxylate terminal group, whereas the method of Akita provides an ester terminal group. Also see S. Coulombe, et al., "High-Pressure Liquid Chromatography for Fractionating Oligomers from Degraded Poly(.beta.-Hydroxybutyrate)", Macromolecules, 11, pp. 279-280 (1978); and A. Ballistreri, et al., "Sequencing Bacterial Poly(.beta.-Hydroxybutyrate-co-8-hydroxyvalerate) by Partial Methanolysis, High-Performance Liquid Chromatography Fractionation and Fast Atom Bombardment Mass Spectrometry Analysis", Macromolecules, 22, pp. 2107-2111 (1989).
H. Morikawa et al. in Can. J. Chem., 59, pp. 2306-2313, (1981) demonstrated that thermal degradation of a PHA copolyester yields monomeric, oligomeric and polymeric PHAs with olefinic terminal groups. Morikawa et al. pyrolyzed PHB to yield crotonic acid and oligomers of PHB having a terminal crotonate moiety, as shown in the polyester of structural formula (IV). Therefore, pyrolysis of a PHA can provide an ##STR5## oligomer with a reactive vinyl terminal group as a site for further chemical modification of the degraded PHA.
Accordingly, from the above degradation methods, i.e. acidic hydrolysis, alkaline hydrolysis, alcoholysis or pyrolysis, a high molecular weight, biologically-synthesized PHA can be degraded to a relatively low molecular weight PHA that includes one of a variety of reactive terminal functionalities, including hydroxyl, free carboxylic acid, carboxylate, ester, and olefinic functionalities. These reactive terminal functionalities therefore allow the introduction of numerous other types of terminal functionalities onto the degraded PHA polyester.
In the past, interest in PHAs concentrated on their unique biodegradable and biocompatible properties, as well as their various physical properties that range from thermoplastic to elastomeric. The physical and chemical properties inherent to PHAs suggest a variety of applications, such as in controlled drug release systems, biomedical devices, specialty packaging materials, and numerous agricultural applications. However, while PHAs are of general interest because of their biodegradable nature, their actual use as a plastic material has been hampered by their thermal instability. For example, poly-3-hydroxybutyrate is thermoplastic, but also is thermally unstable at temperatures exceeding its melting point of about 180.degree. C. N. Grassie, et al., in Polym. Degrad. Stabil., 6, pp. 47-61 (1984), disclose that a substantial molecular weight reduction of PHB occurs by heating PHB in the temperature range of 180.degree.-200.degree. C. The inherent thermal instability of PHB is partially overcome by incorporating a second monomer unit into the polyester. The melting point of a PHB can, for instance, be reduced to 75.degree. C., as in (P[HBcoHV]) including about 40 mol % 3-hydroxyvalerate, resulting in a polymer that is thermally stable up to about 160.degree. C. However, further enhancements in the thermal stability of PHAs are necessary for their practical use in commercial applications. Also see M. Kunioka, et al., Macromolecules, 23, pp. 1933-1936 (1990).
Accordingly, prior investigators have studied the chemical and biological synthesis of PHAs, and the degradation of PHAs, in attempts to provide a biodegradable polymer having physical and chemical properties suitable for consumer, industrial and agricultural applications. However, the prior investigators have studied essentially only homopolymeric and copolymeric hydroxyalkanoates. In general, to date, very few known references are directed to a compound, or its method of preparation, including a PHA polymer functionalized with a moiety other than a poly(hydroxyalkanoate).
Some investigators, like P. B. Dave et al., in "Survey of Polymer Blends Containing Poly(3-Hydroxybutyrate-co-16% Hydroxyvalerate", in Polymer Preprints, 31(1), pp. 442-443 (1990), studied the physical compatibility of a PHA blended with other commercial polymers. However, these were physical blends of a PHA with a second polymer, like a poly(ethylene oxide), and did not include a PHA polymer covalently attached to a molecule or a polymer other than a PHA. R. I. Hollingsworth et al. in Carbohydrate Research, 134, pp. C7-C11 (1984) and R. I. Hollingsworth et al. in Journal of Bacteriology, 169(7), pp. 3369-3371 (1987) found 3-hydroxybutyrate covalently attached as a noncarbohydrate substituent in the acidic capsular polysaccharide and extracellular polysaccharide of Rhizobium trifolii. However, the 3-hydroxybutyrate substituent was monomeric and was substituted biologically, not chemically. M. S. Reeve et al., in "The Chemical Degradation of Bacterial Polyesters for Use in the Preparation of New Degradable Block Copolymers", Polymer Preprints, 31( 1), pp. 437-438 (1990), disclose a polyurethane-type copolymer derived from the reaction of 4,4'-diphenylmethane diisocyanate with polyethylene glycol and degraded PHB, and disclose a PHB-polystyrene block copolymer derived from degraded PHB and a polystyrene prepolymer including a carboxylic acid functionality.
However, no known prior art reference teaches or suggests a compound including a degraded PHA covalently attached to a polysaccharide or to a carbohydrate to provide a novel class of polymers having improved physical properties. The physical properties demonstrated by the novel, biodegradable polymers of the present invention, such as improved solubility in an aqueous solvent and improved thermal stability, are unique and useful, and are distinguished and improved over the physical properties of both the PHA polyester itself and the non-PHA moiety, or moieties, covalently attached to the PHA polyester. Furthermore, the novel poly(hydroxyalkanoate) derivatives can be designed to possess physical properties intermediate between the physical properties of the individual PHA and non-PHA components of the functionalized poly(hydroxyalkanoate) derivative.